The consistent occurrence of Francisella in both our lab-grown and field-collected tick samples suggests its systemic association with ticks. Our analysis has assigned abundant sequences to Francisella (70–75%), followed by Midichloria (14–17%) and Rickettsia (6–9%), excluding unfed ovary tissue. The richness of Francisella in immature and mature life developmental stages and individually-dissected tissues (except for the unfed ovary) supports the notion that it sustains an obligatory association with all feeding stages and outcompetes other bacteria. The relative abundance of Midichloria and Rickettsia was considerably lower than Francisella, signifying their link with the tick as facultative endosymbionts. However, competitive interactions among endosymbionts, pathogenic microbes, have been shown to increase virulence of pathogenic microbes, and complications in vertical transmission were suggested to influence obligatory or facultative endosymbionts in ticks . Consistent dominance of Francisella in tick life stages and tissues suggests its significance in Am. americanum development and other biological processes. Perhaps the Am. americanum ticks are sheltering Francisella by creating a favorable niche within various tissues vector for the colonization, propagation, and trafficking of Francisella at the expense of other endosymbionts and microbial communities. The tick vector sequestered vital nutrients from Francisella for development, and in return provided the niche for Francisella to survive and thrive within the tick vector. Synergistic and antagonistic interactions among pathogenic and non-pathogenic rickettsial species could also play an important role in the prevalence of microbes within the tick vector. The interspecies competition among Midichloria and Rickettsial species could also cause their decreased prevalence within the arthropod vector. For example, dominant Rickettsia peacockii blocked vertical transmission of R. rickettsii in the ovary of Dermacentor andersoni [44–45]). The dominance of Francisella-like endosymbionts (FLEs) in Am. americanum might be location-specific, as demonstrated for Coxiella and Rickettsia [46–47], and of fundamental importance because of its occurrence in several other tick species such as Dermacentor occidentalis Marx, 1892, Dermacentor albipictus (Packard), Dermacentor nitens Neumann, 1897, D. andersoni Stiles, 1908, Dermacentor hunteri Bishopp, 1912, and Dermacentor variabilis (Say, 1821) [45–49]. Still, the nature of their symbiotic relationship is yet to be explained. In this study, field-collected tick tissues from Illinois, Delaware, and Maryland also demonstrated the presence and dominance of FLEs, suggesting their establishment in field populations of Am. americanum ticks, although their presence seems much prominent in lab-grown colonies (Fig. 10).
We found differences in the diversity metrics between lab-raised and field-collected ticks colonies. The observed_OTUs metrics, a quantitative measure of the total number of bacteria, showed that field-collected ticks have about 100 folds more bacteria than lab-raised ticks irrespective of sample type (Fig. S1). We also found that field-collected ticks have a higher diverse microbiome regardless of the tick location than lab-raised tick colonies. To further strengthen this observation, despite the fact Francisellaceae was the dominant bacterial family in both tick groups, field-collected ticks also harbored other bacterial families at a relatively high abundance contributing to a more diverse microbiome. This finding implies that the tick environment plays a significant role in enriching certain bacteria members of the tick microbiome. The lab-raised ticks have been maintained under strict laboratory conditions with limited vertebrate hosts and have less richness in their microbial communities. It seems possible that the differences in the diversity observed amongst life stages of field-collected and lab-raised ticks is due solely to the continuous exposure to environmental bacteria encountered by questing ticks in the field. A closer look at bacterial profile in Figs. 2 and 3 clearly indicates that with the exception of the presence of Francisellaceae in both laborotory raised and field-collected ticks, several bacterial family were exclusively detected in field-collected ticks such as Propionibacteriaceae, Nocardiaceae, Dermabacteraceae, Rickettsiales and Staphylococcaceae all of which are known to be environmental residents in soils and are predominant on both human and animal skin. These findings provide further support for the hypothesis that the tick microbiome may play a contributing factor to the incidence of the alpha-gal phenomenon across different region in the United States.
An earlier study conducted on field-collected Am. americanum ticks from Indiana showed ~ 40% of Coxiella-like endosymbionts, followed by ~ 5% rickettsial endosymbionts . However, ticks collected from North Carolina harbored a preponderance of Rickettsia-like endosymbionts over Coxiella . In the current study, the most abundant bacterial family is Francisella at the organismal level in both immature and adult tick life stages and individually dissected tissues (excluding unfed ovary), suggesting that the microbiome composition of this tick varies with geographical distribution. The presence of Coxiella in unfed ovary tissue indicates its obligate symbiont nature, and another study has shown its presence in the ovary (to show its maternal transmission) and Malpighian tubules to suggest its possible role in osmoregulation and excretion [7, 22–23]. Surprisingly, CLE was neither detected in tick developmental life stages nor tissue samples, except in the unfed ovary. CLE, an obligatory endosymbiont, is required for tick survival and reproductive fitness . CLE has been implicated for its possible role in synthesizing amino acids and several B vitamins [7, 22–23]. Possible reasons for the replacement of CLE in Am. americanum could be the acquisition of FLE and rickettsiae established as alternative obligate symbionts in some tick species . All genes required for vitamin B9 and B7 biosynthesis are also present in Rickettsia and FLE endosymbionts, respectively. In instances where multiple endosymbionts provide a similar advantage to the host, the presence and maintenance of all endosymbionts are not expected; it does not add additional help to the tick host .
It was a common understanding that hematophagous niches are commonly driven by evolutionarily stable symbiotic interactions in several arthropods [52–53]. Contrary to that, an elegant study proposed that obligate symbioses are relatively unstable in obligate hematophagous ticks . In that study, six genera of distinct endosymbionts were present in the castor bean tick (Ixodes ricinus) and the African blue tick (Rhipicephalus decoloratus) but no symbiotic community structure was found fixed and stable across the tick phylogeny . The success of horizontal and vertical transmission patterns in ticks could also modify symbiotic interactions . This fact could also explain why there is low evolutionary stability of the symbiosis between Am. americanum ticks and CLE or CLEs are missing from most of our tick samples in the current study.
CLE follows two different evolutionary strategies. Some CLEs are highly specialized to the tick host from ancient times, followed by co-diversification. For example, the Rhipicephalus genus and CLE lineages have co-diversified together. The emergence of the Rhipicephalus genus and original CLE infection co-occurred, ~ 14 million years ago [25, 54]. On the other hand, other CLEs appear to be acquired through horizontal transfer (HT) from unrelated or accidental host species. Such a pattern has also been observed in other endosymbionts such as Wolbachia, which occurs frequently in insects, similar to CLE in ticks. A recent study also supports the hypothesis of frequent replacement of obligate symbiont CLE in ticks . Our data provide strong evidence that CLE replacement by FLE occurs in Am. americanum populations; otherwise, FLEs are rare in ticks and not frequently found in arthropods [25–26, 45, 49, 55–56]
Earlier studies proposed that among Amblyomma tick species, CLEs have reduced genomes, a feature associated with microbes that are long-term or early evolved endosymbionts . A wide distribution of genetically differentiated strains of CLE across the tick phylogeny also specifies its ancient symbiotic relation with ticks . Unlike the highly reduced genome of CLEs, FLEs have minimal genome reduction and evolved recently from a mammalian pathogen Francisella tularensis  and have more superior biosynthetic metabolic capabilities than CLEs. Therefore, it is highly likely that it has replaced ancestral CLEs with reduced functionality in Am. americanum developmental stages and tissue samples, as evident from the data presented here. A recent study has also suggested replacement of endosymbiont CLEs with FLEs in another Amblyomma tick species, the Gulf Coast tick (Amblyomma maculatum), for the same reason . It is also possible that Am. americanum replaced CLE at the expense of functionally important symbiotic genes (via lateral gene transfer), with FLEs which are functionally more efficient than CLEs because CLE has a reduced genome and is less efficient in metabolic and biosynthetic capabilities, including that of B vitamin synthesis. Perhaps, this is the key evolutionary mechanism of how ticks retain their capacity to synthesize vitamin B even without containing endosymbionts. Previously, this pattern has been reported from some filarial nematodes, which demonstrated the ability to live and reproduce without obligate symbionts through lateral gene transfer . A close examination of Ixodes scapularis, a tick species deficient in CLE, did not show any sign or evidence of lateral gene transfer . However, it contained a rickettsial endosymbiont, Rickettsia buchneri, which could synthesize the B9 vitamin . The presence of Rickettsia buchneri provides a possible explanation for both the absence of CLE and no evidence of lateral gene transfer. An unexplored ecological pathway area facilitating the horizontal transfer (HT) of endosymbionts among tick species needs to be thoroughly investigated. Co-feeding of different tick species on a shared vertebrate host could be an important determinant. Reports have also shown that the salivary glands of blood-fed ticks contain high levels of CLE in some tick species [7, 18]. Thus, the co-feeding of tick species on a shared vertebrate host could serve as an ecological arena for exchanging endosymbionts. Earlier studies have suggested that tick pathogens also colonize with closely related endosymbionts, and these endosymbionts also appear to promote closely related pathogen acquisition and transmission . The FLEs are known to have evolved from the pathogen Francisella tularensis (Ft); however, unlike virulent Ft, its transmission and virulence in humans are enigmas [61–62]. A wide distribution of FLEs in other tick species has been further highlighted by the reports such as > 94% of FLE positives among D. variabilis, D. andersoni and D. occidentalis ticks in the western United States [45, 63], 41% FLE-positives in D. occidentalis ticks from California (Western United States) but without Ft infection . In another earlier study, D. andersoni ticks collected from Oregon and Montana (northwestern United States) showed FLE and Ft accounted for 80% (60% FLEs and 20% Ft) of midgut microbiome , suggesting that genetic similarity of FLE and Ft, and geography are both probably contributing to inflating Ft infection rates. More studies are needed to clarify the involvement of FLEs in Ft infection and how factors such as geography and genetic similarity are involved.
Our network analysis showed a strong positive correlation between Rickettsiaceae and Midichloriaceae families in lab-raised tick life-stages and between Francisellaceae, Rickettsiaceae, and Midichloriaceae in tissues isolated from lab-raised ticks. Interestingly, the presence of the Rickettsiaceae family was shown to be positively correlated to Midichloriaceae across different life-stages of lab-raised tick colonies, indicative of a potential synergistic relationship between bacteria belonging from these two families. This observation was also reported in another study . It showed the Rickettsia parkeri colonization of the tick tissue facilitates replication of the endosymbiont Candidatus Midichloria mitochondrii (CMM) in Am. maculatum ticks. Lejal et al.  also identified a substantial prevalence of CMM in Ixodes ricinus ticks that were positive with bacteria in the Rickettsia genus.
The Coxiellaceae is replaced with the Francisellaceae in Am. americanum ticks and this pattern was further strengthened by analyzing the correlations between bacterial families identified in tissues of lab-raised tick colonies. While we observed no direct correlation among bacteria in the Coxiellaceae family and Rickettsiaceae, Francisellaceae, and Midichloriaceae, the Coxiellaceae family was positively correlated with Rhizobiaceae and Xanthobacteraceae, both of which were negatively correlated to Rickettsiaceae, Francisellaceae, and Midichloriaceae bacterial families. Both Rhizobiaceae and Xanthobacteraceae are non-resident, opportunistic bacteria that ticks acquire from their environment [17, 46–67]. Lejal and colleagues  detected a significant abundance of a Rhizobiaceae-Multi_1 in Rickettsia-positive Ixodes ricinus ticks, suggesting a completely different observation of our findings that Rhizobiaceae was negatively correlated with the Rickettsiaceae, but positively correlated with Coxiellaceae. This finding could present a potential interaction between known bacterial endosymbionts and possible environmental bacteria transiently acquired by ticks. An understanding of how these environmental microbes change the dynamics of the tick microbiome requires further attention.
The presence of specific microbes in tick salivary glands is vital in the context of α-gal syndrome (AGS) as tick bites are believed to be responsible for causing AGS in humans. The lone star tick possesses α-gal antigens in its salivary gland and saliva which are hypothesized to be prime culprits of AGS . The gene that codes for the enzyme α-1,3-galactosyltransferase, has not been identified in a tick so far, indicating that the microbiome of the tick salivary gland could be one major contributor of α-gal antigens . Several pieces of evidence support this hypothesis. One important tick-borne bacterial pathogen, Anaplasma phagocytophilum, is linked with an increase of alpha-gal antigen . The recent change of guard within Am. americanum (CLE replaced by FLE) points to the potential role of the tick microbiome in the emergence of AGS. The exclusive replacement of the Coxiellaceae with Francisellaceae in both lab-raised and field-collected Am. americanum ticks could shed more light on the role played by this tick in the emergence of AGS. While there are no current studies on when the pathogenic Francisella tularensis switches to an endosymbiont, Gerhart et al  argued that an FLE of Am. maculatum recently evolved to becoming an endosymbiont. Similarly, it is worth noting that the emergence of AGS in humans and the incrimination of Am. americanum in inducing this condition are recent development. Understanding whether these two phenomena took place concurrently or simultaneously will significantly fill a huge gap in our understanding of the microbiome-tick vector interaction in the context of AGS.
An elegant recent study conducted on the human gut microbiome reported gut resident microbes from the Enterobacteriaceae family, including Escherichia coli, Pasteurellaceae genera, Haemophilus influenza, and Lactobacillus species, can exhibit α 1,3-galactosyltransferase activity, which indicates that the presence of an enzyme in these microbes could contribute to the devolvement of an α-gal antigen . This study also identified specific genes exhibiting α-1, 3-galactosyltransferase bacterial sequences in their shotgun sequencing data . An earlier study reported microbes from Rhizobiaceae and Caulobacteriaceae families possessing novel lipid A a-(1,1)-GalA transferase gene(rgtF) . This enzyme could also be a potential source of an α- gal antigen. In this study, we identified microbes from Rhizobiaceae and Caulobacteriaceae family in Am. americanum salivary glands samples. The microbes from these families could contribute to the synthesis of an α-gal antigen or overall α-gal signature in tick salivary glands. The tick microbiome possessing an uncharacterized enzyme with a glycoside hydrolase, glycosyltransferase, or similar function as α-1, 3-galactosyltransferase, is yet to be investigated.